Many clinically relevant proteins are found on cell surfaces or are secreted from cells. Such proteins include, but are not limited to, peptide hormones, cytokines, lymphokines, cell surface antigens, cell adhesion molecules (CAMs), and homing receptors. They represent a small percentage of all proteins synthesized by cells. Such proteins are usually identified initially on the basis of their activity, and then intensive scientific investigation must be carried out in an effort to isolate and characterize the proteins. Once such proteins are isolated, the genes encoding them may be isolated and cloned by a variety of methods for use in large-scale protein production. Such cloning methods require at least partially purified protein and additional information about the protein such as amino acid residue sequence, activity or antibodies specific for the protein.
In one cloning strategy, the protein is isolated, a partial amino acid residue sequence of the protein is determined, and a set of degenerate nucleic acid probes is designed based on the amino acid sequence. The probes are then used to screen DNA libraries. This approach is suitable for use where only small amounts of protein can be isolated to a high degree of purity, and where an amino acid sequence can be obtained. Where larger amounts of purified protein are available, antibodies to the protein may be made and used to screen so-called expression DNA libraries. These expression libraries utilize hybrid genes in which each cDNA is fused to a gene fragment encoding a signal peptide, so that the expressed protein is secreted by virtue of the exogenous signal peptide. The secreted proteins are then screened by the antibodies specific for the secreted protein. This approach may be suitable for use where the protein can be obtained in sufficient purity and amounts for obtaining antibodies.
DNA libraries are obtained either from the complete genomic DNA, a random fraction of the complete genomic DNA, or from cDNA derived from total cellular mRNA. Genes encoding cell surface and secreted proteins account for only a small fraction of genes encoding the bulk of proteins. Therefore, efforts at cloning cell surface and secreted proteins are laborious, time-consuming and not possible without having first obtained the protein or some knowledge about the protein.